Summary of work: Insulin elicits pleiotropic effects through the insulin receptor (IR), whose intrinsic tyrosine kinase activity is required for its function. The carboxyl-terminal domain is one of several structural regions that has been defined within the intracellular portion of the insulin receptor beta-subunit to contribute to the propagation of the insulin signal. In this study, the functional importance of the carboxyl-terminal domain of IR was ascertained by stable expression of a peptide corresponding to residues 1293-1307 of this receptor domain in Chinese hamster ovary cells harboring large number of IR. The ligand-mediated responses of a number of parameters were analyzed in these cells, including receptor phosphorylation, thymidine incorporation into DNA, phosphatidylinositol 3'-kinase activity and mitogen-activated protein kinase activation. The peptide expression did not influence IR expression. While having minimal effect on basal responses, a significant 2-fold enhancement of insulin-stimulated IR activity and signaling was observed in several peptide-expressing cellular clones when compared to cells transfected with the vector alone. Under these experimental conditions, receptor phosphorylation and DNA synthesis in response to insulin-like growth factor 1 remained unaffected. Thus, a discrete non-catalytic region of the IR carboxyl-terminal domain appears to be involved in the stimulation of insulin receptor signal transduction in response to insulin. Importantly, the minigene approach represents a useful method for expressing receptor cytoplasmic domains that can modulate IR functions in cells.